Physicians and veterinarians often prescribe antibiotics for their sick patients on the basis of antibiotic interaction tests that presumably determine the inhibitory activity of antibiotics against the infectious microorganism. However, there can be significant problems. Sometimes a prescribed antibiotic fails to cure an infection even though the pathogen is reportedly susceptible. In serious infections, this failure can be fatal.
There can be several reasons why prescribed antibiotics fail to work, such as patient involvement, pathogen-related factors, and, most remarkably, a deficiency in the antibiotic interaction test used by the physician in selecting the appropriate antibiotic. The deficiency in the antibiotic interaction test is as follows: the test fails to account for the inactivating potential of some microorganisms toward particular antibiotics.
It is common knowledge that certain bacterial enzymes can inactivate particular antibiotics. Most notably, the β-lactamase enzymes inactivate β-lactam antibiotics, which include the most commonly prescribed antibiotics such as penicillins, cephalosporins, cephamycins, monobactams, monocarbams, penems or carbapenems (Bauernfeind et al., (1989) “Extended broad-spectrum β-lactamase in Klebsiella pneumoniae including resistance to cephamycins” Infection 17:316-321.) In choosing an effective antibiotic therapy, consideration of the presence of β-lactamase enzymes is crucial because they have been reported in infectious bacterial isolates of E. coli, K. pneumoniae, K. oxytoca, Salmonella spp., Citrobacter freundii, Enterobacter aerogenes, and Proteus mirabilis, to name a few. However, β-lactamase enzymes are not reliably detected in current antibiotic interaction tests. Therefore, the use of such flawed tests may lead to treatment failure and, ultimately, the resurgence of the infection in the sick patient.
At the present time, there are two conventional types of antibiotic interaction tests, disk diffusion methods or antibiotic dilution methods. In disk diffusion methods, a standard quantity of the infectious microorganism is uniformly spread over the surface of an appropriate culture medium (hereafter referred to as agar), then several filter paper disks impregnated with specific amounts of selected antibiotics are placed on the agar surface (for example see Bauer et al., (1966) Am. J. Clin. Path. 45:493-496; Bell, (1975) Pathology 7:Suppl 1-48; Stokes et al., (1972) Association of Clinical Pathologists Broadsheet, No. 55 (revised)). During incubation, the microorganism grows on the surface of the agar except in the areas where certain antibiotics have inhibited its growth. Inhibition of growth is detected as clear zones of no growth (inhibition zones) on the agar around the specific antibiotic disk. The sizes of the inhibition zones are measured and compared to determine the microorganism's interaction to the particular antibiotic.
On the other hand, in the dilution method, a constant quantity of microbial inoculum is introduced into a series of tubes or wells of broth containing varying concentrations of antibiotic [NCCLS (1997) “Methods for dilution antimicrobial interaction tests for bacteria that grow aerobically” Approved standard M7-A4, National Committee for Clinical Laboratory Standards, Villanova, Pa.]. After incubation, the broth tests are inspected and the lowest concentration of antibiotic that prevents detectable growth of the microorganism is recorded. This concentration is the minimum inhibitory concentration (MIC) of the antibiotic. Neither the disk diffusion nor the dilution methods provide any information regarding the presence of antibiotic-inactivating enzymes in the bacterial culture.
However, various techniques for detecting the presence of bacterial enzymes have been reported. For example, specific tests for the detection of chloramphenicol acetyltransferase, an enzyme that inactivates chloramphenicol, have been developed [Chauchereau et al., (1990) Anal. Biochem. 188:310-316]. These complex tests require special instruments capable of measuring the absorbance of light at specific wavelengths and/or the presence of radioactive labels. Such tests are not antibiotic interaction tests and their complexity is such that they are unsuitable for routine clinical microbiology laboratories.
A similar technique is found in the detection of the antibiotic activity of a bacterial enzyme by the production of a distinctive heaped-up margin of the inhibition zone around a penicillin antibiotic disk from the β-lactamases of Staphylococcus aureus [Gill et al., (1981) J. Clin. Microbiol. 14:437-40]. Bacterial β-lactamase production can also be detected chemically by testing the bacteria with an indicator substance such as nitrocefin [Oberhofer et al., (1982) J. Clin. Microbiol. 15:196-199; O'Callaghan et al., (1972) Antimicrob. Agents Chemother. 1:283-288]. These tests are indicators only of β-lactamase-determined resistance of Staphylococcus aureus, Staphylococcus epidermidis, Moraxella catarrhalis, Neisseria and Haemophilus species to certain types of penicillin antibiotics. They do not predict the potential for any other bacteria to resist these penicillins, nor do they predict the potential for any bacteria to be resistant to any of the other classes of β-lactam antibiotics, such as cephalosporins, cephamycins, monobactams, monocarbams, penems or carbapenems. In short, these are useful tests of limited scope. For tests of β-lactam antibiotics, a more comprehensive test is needed to detect the activities of all β-lactamases against all β-lactam antibiotics.
In another technique, disk diffusion tests were modified by a pre-incubation procedure to determine the ability of β-lactamases from Staphylococcus aureus to inactivate β-lactam antibiotics [Lacey et al., (1977) J. Clin. Pathol. 30:35-39]. This procedure results in smaller inhibition zones than those for which the interpretive criteria of the tests were calibrated. The preincubation procedure thereby invalidates the interpretive tables that are necessary to determine antibiotic interaction or resistance. This is a serious deficiency because it would be unethical to base therapy on this procedure that lacks validated interpretive criteria.
Another test is the clover leaf test which has been used to detect β-lactamases and is also claimed to detect two other types of antibiotic-inactivating enzymes, chloramphenicol acetyltransferase and erythromycin esterase [Andremont et al., (1982) “Proceedings Reunion Interdisciplinaire de Chimiotherapie Antiinfectieuse” Societe Francaise de Microbiologie, Paris:50; Kjellander et al., (1964) Acta Path. Microbiol. Scand. 61:494.] This test is not an antibiotic interaction test and must be set up as an additional procedure, a disadvantage for routine laboratory testing. Furthermore, there are doubts about the validity of results obtained with this procedure [Jorgensen (1985) Chemotherapy 31:95-101; Reig et al., (1984) E. J. Clin. Microbiol. 3:561-562].
Still another technique includes the cefoxitin induction test [Sanders et al., (1979) Antimicrob. Agents Chemother. 15:792-797] for detecting a particular type of bacterial β-lactamase, the inducible AmpC β-lactamase of Bush Group 1 [Bush et al., (1995) AAC 39:1211-1233]. This test does not detect all types of β-lactamases, and like the clover leaf test, it is a specialized and complicated procedure used to supplement antibiotic interaction tests.
Yet another technique is the double disk potentiation test which involves strategically placing an amoxicillin/clavulanate or ticarcillin-clavulanate disk at a distance of about 20 to 30 mm from disks containing cefotaxime, ceftriaxone, ceftizoxime, ceftazidime, cefepime or aztreonam on an agar plate. It is therefore possible to determine if a strain of Enterobacteriaceae produces a special type of β-lactamase known as an extended-spectrum β-lactamase [Brun-Buisson et al., (1987) Lancet ii:302-306]. The test is based on the ability of the β-lactamase inhibitor, clavulanate, to inhibit the extended-spectrum β-lactamase and prevent it from inactivating the cephalosporin or aztreonam antibiotics in the test. This is a special procedure, not a routine antibiotic interaction test, and detects only certain types of β-lactamases. It is therefore inconvenient and limited in scope.
A variety of disk and dilution tests have been derived from the principle of the double disk test [Brown et al., (2000) J. Antimicrob. Chemother. 46:327-328; Cormican et al., (1996) JCM 34:1880-1884; Ho et al., (1998) J. Antimicrob. Chemother. 42:49-54; Moland et al., (1998) J. Clin. Microbiol. 36:2575-2579; Sanders et al., (1996) J. Clin. Microbiol. 34:2997-3001; Schooneveldt et al., (1998) Pathology 30:164-168; Thomson et al., (1999) Antimicrob. Agents. Chemother. 43:1393-1400]. That is, they use the ability of a β-lactamase inhibitor to inhibit an extended-spectrum β-lactamase to detect this type of β-lactamase.
In another technique, the direct 3-dimensional test, [Thomson et al., (1984) J. Antimicrob. Chemother. 13:45-54; Thomson et al., (1992) AAC 36:1877-1882; U.S. Pat. No. 5,466,583], a standard quantity of the causative microorganism is uniformly spread over the surface of an agar plate in the usual manner for performing a disk diffusion test. However, before placing the antibiotic disks onto the surface of the agar, the 3-dimensional inoculation is performed. This is effected by using a sterile scalpel to cut a slit in the agar about 3 mm to one side of where the antibiotic disks will be placed. A dense liquid inoculum of the test microorganism is then dispensed into the slit, the antibiotic disks are placed on the agar 3 mm from the slit, and the test is incubated.
After incubation the inhibition zones are measured by standard procedures to determine the interaction of the microorganism to the test antibiotics according to the interpretive criteria of the disk diffusion test. However, in addition to this, enzymatic inactivation of the antibiotics can be detected by inspecting the intersections of the 3-dimensional inoculum with the margins of the inhibition zones. Antibiotic inactivation results in a distortion or discontinuity in the usually circular inhibition zone. (These distortions or discontinuities are hereafter referred to as “3-dimensional effects”.)
The 3-dimensional test thus permits the laboratory to report to the clinician not only the interaction or resistance of a microorganism to antibiotics, but also the ability of the microorganism to inactivate the antibiotics. As a hypothetical example, whereas a conventional antibiotic interaction test might indicate that a microorganism was susceptible to the two antibiotics, cefaclor and cefoxitin, the 3-dimensional test can provide additional information to show that the microorganism produced an enzyme capable of inactivating cefaclor but not cefoxitin. Thus, although the conventional test indicated that both antibiotics appeared to be equally efficacious, it would appear, from the additional information provided by the 3-dimensional test, that only cefoxitin might not be inactivated in the patient and therefore would constitute a more effective treatment than cefaclor. In this example, the information provided by the 3-dimensional test could assist a clinician to make a better choice of therapy.
In addition to the direct form of the 3-dimensional test, an indirect form is used for testing microorganisms when inhibition zones are small or absent, or as a research or diagnostic method. The indirect test is performed by inoculating the surface of the agar with a fully susceptible assay strain such as Escherichia coli ATCC 25922. After this, the 3-dimensional slit is cut in the agar and inoculated with a suspension of the test microorganism. Although the indirect test precludes the simultaneous determination of antibiotic susceptibilities, it permits investigation of the antibiotic inactivating enzymes of microorganisms for which the inhibition zones are too small to yield 3-dimensional results when the test is performed in the direct form of the test.
There are several problems with the 3-dimensional test. These problems include the following:                a) The procedure for making the slit in the agar for the 3-dimensional test is inconvenient and technically difficult to perform correctly;        B) Making the slit is potentially dangerous to laboratory staff because a scalpel blade contaminated with pathogenic bacteria is an infection hazard; and        c) It is also technically difficult to accurately deliver the liquid 3-dimensional inoculum into the slit without overfilling the slit and possibly invalidating the test.        
As disclosed hereinafter, the present invention provides one solution to the problems of current antibiotic interaction tests used by the physician in selecting the appropriate antibiotic. The present invention provides an effective method for evaluating the inactivating potential of some microorganisms toward particular antibiotics.